SEQUENCE, GENETIC, AND LACZ FUSION ANALYSES OF A NIFR3-NTRB-NTRC OPERON IN RHODOBACTER-CAPSULATUS

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are glnB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus. nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli regulatory gene, fis. Analysis of ntrC-lacZ fusions and complementation data indicate that nifR3 ntrBC constitute a single operon. nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription). This gene organization suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R. capsulatus.